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. Author manuscript; available in PMC: 2020 Jan 1.
Published in final edited form as: Mol Cancer Res. 2018 Sep 13;17(1):30–41. doi: 10.1158/1541-7786.MCR-18-0246

Figure 3. Restoration of miR-200c abrogates expression of immunomodulatory gene and protein expression in TNBC cells.

Figure 3.

(A) Relative TDO2 mRNA levels in BT549-TripZ-200c cells after 72 hours of no treatment, DOX, NFκB stimulation (TNF-α, IL-1β (10 ng/μL each)) for the last 48 hrs, or the combination of DOX and NFκB stimulation (top). Relative TDO2 levels were normalized to β-actin. ***P<0.001, **P<0.01 by unpaired t-test. Immunoblot for TDO2 and ZEB1with α-Tubulin as a loading control in BT549-TripZ-200c cells after 72 hours of no treatment, DOX, NFκB stimulation (TNFα and IL-1β), or the combination of DOX and NFκB stimulation (bottom). Fold changes are indicated compared to vehicle treated after normalizing to loading control.(B) IL6 mRNA levels in BT549-TripZ-200c cells after 72 hours of no treatment, DOX, NFκB stimulation(TNF-α, IL-1β), or the combination of DOX and NFκB activation. Relative IL6 levels were normalized to B-actin. ***P<0.001, **P<0.01 by unpaired t-test. (C) Relative miR-200c in BT549-TripZ-200c cells after 72 hours of no treatment, DOX alone, NFκB stimulation (TNF-α, IL-1β), or the combination of DOX plus NFκB activation. Relative miR-200c levels were normalized to U6 snRNA. (D) HMOX-1 qRT-PCR in multiple TNBC cell lines following transient transfection with either negative control scrambled (SCR) mimic or miR-200c mimic after 48 hours (left) and immunoblot for HO-1 (encoded by HMOX-1) in TNBC lines following transient transfection with SCR mimic or miR-200c mimic for 72 hours (right). Fold changes were calculated over SCR after normalizing to the α-Tubulin loading control. (E) Immunoblot for HO-1 protein in in BT549-Tripz-EV cells as compared to BT549-Tripz-200c cells treated minus or plus DOX (72 hrs) with or without TNF-α for the last 48 hrs. (F) Immunoblot for GDF-15 in MDA-453 cells following transfection with SCR control or miR-200c mimic for 72 hrs with or without NFκB stimulation for the last 48 hrs (left) and in BT549-TripZ-200c cells after vehicle versus DOX for 72 hrs with or without NFκB stimulation for the last 48 hrs (right). Fold change was calculated over vehicle treated cells after normalization to α-Tubulin loading control.